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EP0350273B1 - Procédé pour la détermination d'endotoxines - Google Patents

Procédé pour la détermination d'endotoxines Download PDF

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Publication number
EP0350273B1
EP0350273B1 EP89306795A EP89306795A EP0350273B1 EP 0350273 B1 EP0350273 B1 EP 0350273B1 EP 89306795 A EP89306795 A EP 89306795A EP 89306795 A EP89306795 A EP 89306795A EP 0350273 B1 EP0350273 B1 EP 0350273B1
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EP
European Patent Office
Prior art keywords
endotoxin
concentration
spiked
concentrations
test sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
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EP89306795A
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German (de)
English (en)
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EP0350273A3 (en
EP0350273A2 (fr
Inventor
Masakazu Tsuchiya
Haruki Oishi
Aya Takaoka
Shuji Matsuura
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Fujifilm Wako Pure Chemical Corp
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Wako Pure Chemical Industries Ltd
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Publication of EP0350273A3 publication Critical patent/EP0350273A3/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia

Definitions

  • This invention relates to a process for measuring the concentration of endotoxin (hereinafter abbreviated as "ET”) in a test sample by measuring an optical change due to a gelation reaction caused by reacting horseshoe crab hemocyte lysate (hereinafter abbreviated as "AL solution”) with ET, which process can be employed even when there is a fear that the test sample may affects the gelation reaction caused by the reaction of AL solution with ET.
  • ET endotoxin
  • ET's are known as typical pyrogens. It is known that when blood, an infusion or an injection, which is contaminated by ET, is injected into a body, the ET produces serious side effects such as fever, shock, etc. Therefore, it is considered indispensable to prevent troubles by measuring the amount of ET in starting materials, starting waters, etc. for infusions, injections and the like, or various preparations which are injected into a living body.
  • AL solution has a property of being activated by ET to cause gelation reaction, and a simple, low-cost ET detecting method utilizing this property, i.e., the so-called Limulus test, is widely employed in the fields of medical science, pharmacy and microbiology.
  • the method of Limulus test has been variously improved, and in place of semi-quantitative method according to gel-clat technique which was employed at the beginning, an optical measuring method has come to be employed generally. As a result, more precise determination has become possible.
  • the optical measuring method comprises noting a turbidity due to a gel formed by the gelation reaction of AL solution with ET, measuring the change of turbidity as an optical change, for example, a change of the amount of transmitted light, and determining ET from the change of the amount of transmitted light.
  • the ET concentration in a test sample is determined, for example, from a graph showing the calibration relation (a calibration curve) obtained from a time required for the ratio of transmittance to reach a predetermined value (hereinafter abbreviated as "gelation time") and ET concentration.
  • the above measuring method employed at present can be employed without a particular trouble for measuring the concentration of ET contained in water itself such as starting water, wash water, etc. or for measuring the ET concentration in a test sample which does not affect the gelation reaction of AL solution with ET, it is inapplicable to a test sample which has an inhibitory or enhancement effect on the gelation reaction.
  • a test sample which inhibits the gelation reaction the gelation time is increased, resulting in a measured value lower than a real value.
  • the gelation time is reduced, resulting in a measured value higher than a real value.
  • the amount of ET in a test sample is measured using a calibration curve showing the relationship between ET concentration and gelation time which is obtained using, as samples, solutions of predetermined amounts of ET spiked in distilled water (hereinafter abbreviated as "water calibration curve”), and a calibration curve showing the relationship between ET concentration and gelation time which is prepared using three or more samples prepared by spiking predetermined amounts of ET to identical preparations of a test sample (hereinafter abbreviated as "test sample calibration curve").
  • the calibration curve showing the relationship between ET concentration and gelation time which is obtained using samples prepared by spiking predetermined amount of ET to identical preparations of a test sample containing no ET originally, coincides with the water calibration curve when the test sample does not affect the gelation reaction at all; the former is above the latter when the test sample inhibits the gelation reaction; the former is below the latter when the test sample enhances the gelation reaction; and in most of the cases, the former calibration curve is originally parallel to the water calibration curve.
  • the test sample calibration curve has a portion not parallel to the water calibration curve is that the test sample calibration curve undergoes some influence of ET originally contained in the test sample, and therefore the ET content of the test sample can be measured by comparing the test sample calibration curve with the water calibration curve.
  • the ET content of the test sample cannot be measured unless as samples prepared by spiking ET to identical preparations of test sample, there are prepared at least one sample containing spiked ET in an amount substantially equal to or smaller than the amount of ET originally present in the test sample, and two or more samples containing spiked ET in such amounts that the amount of ET originally present in the test sample can be neglected. Accordingly, said process is disadvantageous, for example, in that the ET content of the test sample should be predicted to a certain degree and that procedures such as the preparation of the samples are troublesome. Thus, further improvement of said process is desirable.
  • This invention was made in consideration of such conditions and is intended to provide a process which permits simple and rapid measurement of the ET concentration in a test sample which tends to affect the gelation of AL solution by ET.
  • This invention provides a process for measuring an endotoxin concentration in a test sample by measuring optical changes due to a gelation reaction caused by a reaction of a horseshoe crab hemocyte lysate with endotoxin, which comprises
  • Fig. 1 shows a calibration curve obtained in Example 1.
  • Fig. 2 is a graph showing the relationship between the concentration of spiked ET (E s ) and apparent ET concentration (E app ) which was obtained in Example 1.
  • Fig. 3 is a graph showing the relationship between E s and E app which was obtained by use of a dextran solution as sample in Example 2.
  • Fig. 4 is a graph showing the relationship between E s and E app which was obtained by use of 20-fold diluted GIT medium in Example 3.
  • Fig. 5 is a perspective view of outer appearance of one example of the apparatus of this invention.
  • Fig. 6 is a block diagram showing an electric system of one example of the apparatus of this invention.
  • This invention is conducted, for example, as follows.
  • ET spiked solutions different in ET concentrations are prepared by spiking ET in various concentrations to substantially identical preparations of a solution which neither contains ET nor affects a gelation reaction caused by reacting AL solution with ET, and each of them is reacted with AL solution, after which a calibration curve showing the relationship between ET concentration and gelation time (hereinafter abbreviated as "ET calibration curve") is prepared.
  • ET calibration curve showing the relationship between ET concentration and gelation time
  • the apparent ET concentration (E app ) of each ET spiked sample prepared from the preparation of test sample is determined.
  • E app a conventional method of statistical straight-line test, for example, a method comprising processing E app and the concentration of actually spiked ET (E s ) statistically and determining a correlation coefficient ( ⁇ )
  • ⁇ a correlation coefficient
  • suitable amounts of ET are spiked to the test sample and the solution containing no ET.
  • the amounts (or concentration) of ET to be spiked are not particularly limited but preferably selected from the range of 0.2 to 20 times, more preferably 0.5 to 10 times, as large as the predetermined endotoxin concentration for judgement depending on purposes (E det ).
  • At least three known different concentrations of ET are spiked to each test sample and each solution containing no ET to give at least 6 sample solutions to be measured. In this case, when E c and E det are quite different, there is a tendency to lower reliability of E c measured.
  • the optical change to reach a predetermined proportion (R th ) is provided in the range of 75 to 97% in terms of R(t).
  • R th when R th is provided at higher than 97%, the moving (convection) in the mixture immediately after mixing the AL solution with ET spiked sample or ET spiked solution, or the variation of R(t) due to the movement of bubbles is possibly judged as gelation by mistake. Therefore, in order to prevent an erroneous detection due to such noises, the range of 3% is necessary for allowance of noise.
  • R th is provided in the range of 75 to 97%, preferably in the range of 80 to 95%.
  • the process of this invention is applicable not only to a test sample which is clearly known to affect the gelation reaction but also to a test sample which is not clearly known to affect and a test sample which does not affect.
  • pyrogen-free distilled water more specifically, distilled water for injection, is usually preferably used, for example, because it is easily available.
  • the AL solution usable in the process of this invention is not critical so long as it can be used for usual measurement of ET.
  • AL solutions prepared from freeze-dried products of AL solutions which are commercially available, for instance, from Associates of Cape Cod Inc. (ACC), HAEMACHEM, Inc., Whittaker Bioproducts, Inc, and Teikoku Hormon Mfg. Co., Ltd.
  • any AL solution without particular restriction so long as it is extracted from hemocytes of horseshoe crab belonging to Limulus genus, Tachypleus genus or Carcinoscorpius genus and reacts with ET to undergo gelation reaction.
  • the measuring process of this invention can easily be practiced by means of an apparatus exclusively for kinetic turbidimetric technique such as Toxinometer ET-201 (mfd. by Wako Pure Chemical Industries, Ltd.), LAL-5000 (mfd. by ACC Inc.), etc., it can be practiced also by means of a measuring apparatus utilizing an optical principle, such as MS-2 of Abbot Laboratries, etc.
  • an apparatus exclusively for kinetic turbidimetric technique such as Toxinometer ET-201 (mfd. by Wako Pure Chemical Industries, Ltd.), LAL-5000 (mfd. by ACC Inc.), etc.
  • an optical principle such as MS-2 of Abbot Laboratries, etc.
  • the present invention also provides an apparatus for measuring endotoxin comprising:
  • a plurality of cuvette holders 7 for holding cuvettes for measurement are provided in one apparatus.
  • Each one optical system for measuring transmitted light amounts is provided in one holder 7. It is also possible to apply a light to each holder by using an optical fiber from one light source. Further, all the holders are kept at a constant temperature such as at 37°C by a constant temperature bath (not shown in Fig. 5) or a dry temperature controlling apparatus.
  • the reaction solution can be maintained at a still state until the end of the measurement and the measurement can be conducted without giving any moving so as to break the gel state.
  • a switch 8 for instructing the beginning of the measurement is instantly pushed to begin the measurement of the sample.
  • the switch 8 is pushed every time when a cuvette for measurement is fixed in a holder 7 in a similar manner to conduct the parallel measurement of a plurality of samples with an optional timing within the number of cuvette holders provided in the apparatus.
  • Fig. 5 shows that it is possible to fix 16 cuvettes in the holders in the apparatus. But the number of cuvettes measurable at a time is not limited to such a number of 16 and can be increased or decreased if desired so long as the capacity for control is permissible. It is also possible to connect switches for instructing the beginning of measurement to each cuvette holder 7. It is further possible to automatically switch on when each cuvette is placed in each cuvette holder so as to instruct the beginning of measurement.
  • a display control switch 12 can be used for changing one after another the display of each T g of each sample when a plurality of samples are measured. It is also possible to display as a whole by using another display method such as CRT display.
  • a keyboard 20 connected to the main body of the apparatus is used for appointment of operational function, input of numeral data, and the like.
  • the display panel 11 is also used for confirming display of input data.
  • Fig. 6 is a block diagram showing an electric system of one example of the apparatus of this invention.
  • the constant temperature controlling system around the cuvettes for measurement is omitted for simplicity and only the optical measurement controlling system is shown.
  • the transmitted light amounts obtained from a plurality of photoelectric detectors 6 are selected one after another successively by a multiplexer 13, converted to R(t) by a ration operating circuit 14, and input into an A/D converter 15.
  • the signal converted to digital amount by the A/D converter is processed by a computer 16 to give the judgement of gelation and the determination of T g .
  • the gelation judgement display 10 and the display panel 11 are also controlled by the computer.
  • the computer 16 prepares and stores calibration curves relating to the gelation time and the ET concentration from the measuring results regarding samples containing ET in known concentrations based on the data input from the keyboard 20. Further, by properly using programs in the computer 16, it is possible to obtain apparent ET concentrations in samples from the gelation times of the samples to which ET is spiked using said calibration curves, to detect linearity between the spiked ET concentrations and the apparent ET concentrations so as to obtain an equation of linear relationship between both concentrations, and to obtain the ET concentration in the sample from a slope and an intercept of the equation of linear relationship. It is also possible to print out the measurement results and the data processing results by a printer 17. It is still possible to connect the computer 16 to one or more outer computers, if required, depending on purposes.
  • this invention provides an apparatus for measuring endotoxin in a plurality of samples in parallel and effectively with high reliability. Further, by using the apparatus of the present invention, the measurement can be carried out precisely even when a test sample includes substances which may affect the gelation reaction caused by the reaction of AL solution with ET.
  • a freeze-dried product of AL solution derived from horseshoe crab belonging to Limulus genus (hereinafter the freeze-dried product being abbreviated as "LAL"; available from Associated of Cape Cod, Inc.; labeled sensitivity 0.03 EU/ml; for dissolution in 5 ml) was dissolved in 5 ml of distilled water for injection, and the LAL solution thus obtained was used as AL solution.
  • LAL freeze-dried product of AL solution derived from horseshoe crab belonging to Limulus genus
  • Solutions having a sorbitol concentration of 2% and a concentration of spiked ET of 0.01, 0.025, 0.05 or 0.1 EU/ml, respectively, were prepared as samples by dissolving sorbitol (mfd. by Wako Pure Chemical Industries, Ltd.; guaranteed reagent) in distilled water for injection to a concentration of 2.2%, followed by adding thereto each of ET solutions having predetermined concentrations.
  • Solutions having a sorbitol concentration of 2% and a concentration of spiked ET of 0.01, 0.025, 0.05 or 0.1 EU/ml, respectively, were prepared as samples by dissolving the same sorbitol as used in i) above in ET solution (0.011 EU/ml) to a concentration of 2.2%, followed by adding thereto each of ET solutions having predetermined concentrations.
  • T g a time required for reducing the transmittance by 5%
  • E s denotes the concentration of spiked ET
  • each value of E app is the average of three measurements.
  • Fig. 2 the relationship between the E app value and the corresponding E s value which was determined for each sample of Group 1 is shown by - ⁇ -.
  • the straight line - ⁇ - in Fig. 2 is a regression line obtained according to individual data by the least squares method.
  • LAL solution obtained by dissolving LAL (available from Associated of Cope Cod. Inc.; labeled sensitivity 0.25 EU/ml; for dissolution in 5 ml) in 5 ml of distilled water for injection.
  • Solutions having a dextran concentration of 5% and a concentration of spiked ET of 0.05, 0.2 or 2.0 EU/ml were prepared as samples by dissolving dextran (available from Wako Pure Chemical Industries, Ltd.; molecular weight 100,000 to 200,000) in distilled water for injection to a concentration of 5.5%, followed by adding thereto each of ET solutions having predetermined concentrations.
  • E s denotes the concentration of spiked ET
  • each value of E app is the average of three measurements.
  • the relationship between the E app values obtained and the corresponding E s values is shown in Fig. 3.
  • the straight line in Fig. 3 is a regression line obtained according to individual data by the least squares method.
  • LAL solution obtained by dissolving LAL (available from Wako Pure Chemical Industries, Ltd.; labeled sensitivity 0.03 EU/ml; for dissolution in 5 ml) in 5 ml of distilled water for injection.
  • Solutions having a concentration of added ET of 0.0125, 0.025, 0.050, 0.10, 0.20 or 0.40 EU/ml, respectively, were prepared as samples by diluting GIT medium (a synthetic medium for cell culture, mfd. by Wako Pure Chemical Industries, Ltd.) in 20 times with distilled water for injection, and adding 0.1 ml of each of ET solutions having predetermined concentrations to 0.9 ml of the diluted medium.
  • GIT medium a synthetic medium for cell culture, mfd. by Wako Pure Chemical Industries, Ltd.
  • E s denotes the concentration of spiked ET
  • each value of E app is the average of three measurements.
  • the relationship between the E app values obtained and the corresponding E s values is shown in Fig. 4.
  • the straight line in Fig. 4 is a regression line obtained by the least squares method.

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Claims (5)

  1. Procédé pour mesurer la concentration d'endotoxine dans un échantillon expérimental en mesurant les changements optiques dus à une réaction de gélification provoquée par la réaction d'un lysat d'hémocytes de limule avec l'endotoxine, dans lequel
    i) on prépare trois échantillons, ou plus, d'endotoxine en injectant de l'endotoxine à différentes concentrations connues , dans un échantillon expérimental,
       on mesure les changements optiques dus à une réaction de gélification provoquée par l'addition du lysat d'hémocytes de limule à des échantillons individuels ayant reçu une injection d'endotoxine, et
       on mesure le temps de gélification nécessaire pour que le changement optique atteigne une proportion prédéterminée,
    ii) on prépare trois ou plusieurs solutions d'endotoxine en injectant de l'endotoxine à différentes concentrations connues, dans une solution ne contenant pas d'endotoxine et n'ayant pas d'influence sur la réaction de gélification,
       on mesure le temps de gélification de la même manière que mentionné en i) ci-dessus, et
       on prépare une courbe d'étalonnage montrant la relation entre le temps de gélification et les concentrations d'endotoxine introduites par injection,
    iii) on obtient les concentrations apparentes d'endotoxine dans les échantillons d'endotoxine à partir des temps de gélification obtenus en i) ci-dessus appliqués à la courbe d'étalonnage obtenue en ii) ci-dessus, et
    iv) on détermine la concentration d'endotoxine dans l'échantillon expérimental en confirmant une linéarité entre les concentrations apparentes d'endotoxine obtenues en iii) ci-dessus et les concentrations effectives d'endotoxine introduites par injection utilisées en i) ci-dessus, et en utilisant un test statistique en ligne droite.
  2. Procédé selon la revendication 1, dans lequel la solution dans laquelle on doit injecter de l'endotoxine est de l'eau distillée injectable.
  3. Procédé selon la revendication 1, dans lequel la concentration d'endotoxine à introduire par injection dans les échantillons expérimentaux ou les solutions est comprise dans un intervalle de 0,2 à 20 fois la concentration prédéterminée d'endotoxine en vue d'une évaluation selon les objectifs recherchés.
  4. Procédé selon la revendication 1, dans lequel le test statistique en ligne droite comprend l'établissement d'une formule de relation linéaire:

    E app = a + b x E s
    Figure imgb0018

    où Eapp est la concentration apparente d'endotoxine; Es est la concentration d'endotoxine effectivement introduite par injection ; et "a" et "b" sont des coefficients, et le calcul de la valeur de a/b.
  5. Appareil pour mesurer l'endotoxine, comprenant
    (a) un bain à température constante pour laisser reposer au moins une cuvette d'échantillon contenant un mélange d'un échantillon expérimental et d'un lysat d'hémocytes de limule dans un état de température constante,
    (b) un moyen pour irradier de la lumière sur les cuvettes d'échantillon individuelles maintenues dans un état de température constante et détecter la quantité de lumière transmise en série,
    (c) un moyen pour indiquer la début de la mesure de la quantité de lumière transmise à travers ladite solution réactionnelle,
    (d) un moyen pour mesurer le temps de gélification nécessaire pour que la quantité de lumière transmise se modifie pour atteindre une proportion prédéterminée à partir du début de la mesure,
    (e) un moyen pour obtenir une équation montrant la relation entre la temps de gélification obtenu en (d) et la concentration d'endotoxine sous la forme de trois échantillons ou plus ayant des concentrations d'endotoxine connues avec des concentration différentes, respectivement, et un moyen pour emmagasinar ladite équation,
    (f) un moyen pour obtenir la concentration apparente d'endotoxine en insérant la temps de gélification obtenu en d) pour trois ou plusieurs échantillons que l'on ajuste en y injectant différentes quantités connues d'endotoxine, dans l'équation obtenue en (e),
    (g) un moyen pour détacter une linéarité entre les concentrations d'endotoxine introduites par injection et les concentrations apparentes d'endotoxine obtenues en (f) pour des échantillons individuels utilisés an (f) et un moyen pour obtenir une équation de relation linéaire entra les deux concentrations, et
    (h) un moyen pour obtenir la concentration d'endotoxine dans l'échantillon expérimental à partir de la pente et de l'ordonnée à l'origine obtenue en (g).
EP89306795A 1988-07-05 1989-07-04 Procédé pour la détermination d'endotoxines Expired - Lifetime EP0350273B1 (fr)

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JP16706888 1988-07-05
JP167068/88 1988-07-05

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EP0350273A2 EP0350273A2 (fr) 1990-01-10
EP0350273A3 EP0350273A3 (en) 1990-10-10
EP0350273B1 true EP0350273B1 (fr) 1994-07-13

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EP (1) EP0350273B1 (fr)
JP (1) JPH07113643B2 (fr)
AT (1) ATE108558T1 (fr)
DE (1) DE68916714T2 (fr)
ES (1) ES2057128T3 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648230A (en) * 1994-06-30 1997-07-15 Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) Endotoxin stabilizing agent, endotoxin composition and method for assaying endotoxin
US20050244299A1 (en) * 2002-04-30 2005-11-03 Biowittaker Technologies Inc Automated sequential injection analysis systems for the determination of trace endotoxin levels
KR101255420B1 (ko) 2008-03-19 2013-04-17 토루 오바타 겔 입자 측정 장치
US9658218B2 (en) * 2012-05-07 2017-05-23 Siemens Healthcare Diagnostics Inc. Determination of total analyte concentration

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62110153A (ja) * 1985-04-03 1987-05-21 Wako Pure Chem Ind Ltd エンドトキシン定量方法

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* Cited by examiner, † Cited by third party
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CA1040081A (fr) * 1974-05-01 1978-10-10 Baxter Travenol Laboratories Methode pour le dosage des endotoxines a l'aide d'un precipite de lysat de limulus
US4038029A (en) * 1976-01-20 1977-07-26 Worthington Biochemical Corporation Limulus lysate turbidity test for pyrogens
ATE76507T1 (de) * 1984-06-27 1992-06-15 Wako Pure Chem Ind Ltd Verfahren zur bestimmung von endotoxin.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62110153A (ja) * 1985-04-03 1987-05-21 Wako Pure Chem Ind Ltd エンドトキシン定量方法

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JPH07113643B2 (ja) 1995-12-06
JPH02124466A (ja) 1990-05-11
DE68916714T2 (de) 1995-01-12
ATE108558T1 (de) 1994-07-15
DE68916714D1 (de) 1994-08-18
ES2057128T3 (es) 1994-10-16
EP0350273A3 (en) 1990-10-10
EP0350273A2 (fr) 1990-01-10

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